Lsrfortesa flow cytometer

For Facs Analysis, differentiated cells were trypsinized, and centrifuged and the pellet was collected in 15 ml tubes, fixed with 100 μl of fixation buffer for 10 min at RT. The fixative was removed by subsequent washing with 1X PBS twice. Permeabilization was performed using 0.1% Triton X for 5–10 min at RT followed by washing twice with 1X PBS. After this, cells were stained with fluorescence-tagged antibody using the stain buffer. For the staining of mesenchymal stem cells, FITC tagged CD73 antibody was used. Cells were incubated with the antibody for 1 h at 37°C. Finally, cells were washed using PBS, transferred in FACS tubes, and analyzed using a BD LSR-FORTESA flow cytometer.

A) PI staining: To determine live and dead cells in response to different reagents, cells were stained with propidium iodide and analyzed by FACS. Cells were treated with different reagents, harvested at 1,200 rpm for 8 min at 4°C, washed with PBS and fixed with 500 μl of 70% ethanol for overnight at 4°C. Cells were then washed with PBS, resuspended in PBS containing 50 μg/ml propidium iodide, 1% triton X-100 and 50 μg/ml RNase A and incubated in dark for 1 h at 37°C. After incubation, cells were centrifuged at 1,200 rpm for 8 min and washed again with PBS, resuspended in sheath fluid and analyzed using BD Biosciences flow cytometer.
B) Annexin V –FITC/PI staining: FACS analysis was also performed to distinguish between live and apoptotic cells by using annexin V-FITC and PI staining (Invitrogen Ltd) as per manufacturer’s instructions. FITC- labelled annexin V binds to membrane phosphatidylserine and propidium iodide binds to cellular DNA. After treatments, cells were washed with PBS and resuspended in 100 μL of 1x annexin binding buffer. Later 5 μL of annexin V and 1 μL of PI was added and incubated for 15 min at room temperature. After incubation, 400 μL 1× binding buffer was added and measured the fluorescence of annexin V-FITC and PI in BD LSRFORTESA flowcytometer. 10,000 events from each sample were acquired to ensure adequate data.

Mice were perfused with PBS, and brains were homogenized in 4 ml of Dulbecco’s PBS (pH 7.4) using Tenbroeck tissue homogenizers. Following centrifugation at 450g for 10 min, cell pellets were resuspended in RPMI containing 25 mM HEPES (pH 7.2), adjusted to 30% Percoll (Sigma) and underlaid with 1 ml of 70% Percoll. Following centrifugation at 800 g for 30 minutes at 4°C, cells were recovered from the 30%-70% interface, washed with RPMI, and suspended in FACS buffer (0.5% bovine serum albumin in Dulbecco’s PBS). Fc receptors were blocked with 1% polyclonal mouse serum and 1% rat anti-mouse CD16/ CD32 (clone 2.4G2; BD Biosciences, San Jose, CA) monoclonal antibody (MAb) for 20 minutes. Specific cell types were identified by staining with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridinin chlorophyll protein (PerCP)-, or allophycocyanin (APC)-conjugated MAb for 30 minutes on ice in FACS buffer. Expression of surface markers was characterized with MAb (all from BD Biosciences except where otherwise indicated) specific for CD45 (clone Ly-5), CD4 (clone GK1.5), CD8 (clone 53–6.7), CD11b (clone M1/70), Ly-6G (clone 1A8), and NK1.1 (clone PK136). Samples were analyzed using a BD LSRFortesa flow cytometer (BD Biosciences) and FlowJo 10 software (Treestar, Inc., Ashland, OR).

Cells were recovered from the 3D tumor matrix by treating the cells with cell recovery solution (Corning), and cell cell suspensions were prepared in PBS with 2% FBS, maintaining a density of 1–5 million cells/mL. Cells were fixed, permeabilized and stained with CD137 (APC) antibody depending on the manufacturers protocol, according to the experimental design. Samples were examined by Accuri C6 Flow Cytometer (San Jose, CA, USA) or BD LSR Fortesa Flow Cytometer (San Jose, CA, USA). Data were processed using FlowJo 10.7.1 software (BD, Ashland, OR, USA).

The analysis of cellular aggresome intensity was performed following the manufacturer’s instructions. The freshly collected cells were stained by PROTEOSTAT® Aggresome detection kit (ENZO, ENZ-51035-K100) and then analyzed by BD LSRFortesa flow cytometer (Wang et al., 2018 (link)).

Cell cycle analyses of live cells that had been stained with 10 mg/ml propidium iodide were conducted using a LSR FORTESA flow cytometer (Becton Dickinson) and the FlowJo software.
Cell cycle synchronization experiments, performed by blocking mouse keratinocytes at the G1/S boundary with a double thymidine block, and cell cycle profiles were analyzed at different time points after release from block.
To analyze cell cycle phases, mouse keratinocytes were infected with lentiviruses expressing the FUCCI sensors (Sakaue-Sawano et al., 2008 (link)). For H2B–EGFP expression, mouse keratinocytes were transiently transfected with a KER14–H2B–EGFP vector (gift from Elaine Fuchs, The Rockefeller University, New York, NY) (Perez-Moreno et al., 2008 (link)).
For cell proliferation analysis, equal numbers of mouse keratinocytes were plated in triplicate. To analyze apoptosis, TUNEL-positive cells were detected using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). For colony formation assays, cells were plated on fibronectin (Merck, New Jersey), fixed 1 week after plating and stained with Rhodamine B.